Next-generation sequencing (NGS) platforms using sequencing-by-synthesis (SBS) technology generate millions of short reads that require bioinformatic assembly. Which step PRECEDES library amplification and is unique to NGS compared to Sanger sequencing?
- A Fragmentation of DNA and adapter ligation to create a sequencing library ✓
- B Dideoxynucleotide chain termination
- C Gel electrophoresis to size-separate fragments
- D Southern hybridization to a labeled probe
Correct answer: A. Fragmentation of DNA and adapter ligation to create a sequencing library
Explanation
In NGS library preparation, genomic DNA is first randomly fragmented (by sonication or enzymatic digestion) to ~150-400 bp fragments, followed by end-repair, A-tailing, and ligation of platform-specific adapter sequences to both ends. These adapters enable bridge amplification (on Illumina flow cells), provide priming sites for sequencing, and include barcodes (indices) for sample multiplexing. Sanger sequencing does not require adapter ligation or high-throughput library prep. Dideoxynucleotide chain termination is specific to Sanger sequencing.
Reference: Harper's Illustrated Biochemistry, 32nd ed.
High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP
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